Desmethylclomipramine triggers mitochondrial damage and death in TGF-ß-induced mesenchymal type of A549 cells.

Lung cancer is the leading cause of cancer deaths, where the metastasis often causes chemodrug resistance and leads to recurrence after treatment. Desmethylclomipramine (DCMI), a bioactive metabolite of clomipramine, shows the therapeutic efficacy with antidepressive agency as well as potential cytostatic effects on lung cancer cells. Here, we demonstrated that DCMI effectively caused transforming growth factor (TGF)-ß1-mediated mesenchymal type of A549 cells to undergo mitochondrial death via myeloid cell leukemia-1 (Mcl-1) suppression and activation of truncated Bid (tBid). TGF-ß1 induced epithelial mesenchymal transition in A549 cells with the increase of fibronectin and decrease of E-cadherin, the activation of Akt/glycogen synthase kinase-3ß (GSK-ß)/Mcl-1 axis, and the hypo-responsiveness to cisplatin. DCMI initiated a dose-dependent cytotoxicity on TGF-ß1-mediated mesenchymal type of A549 cells through inactivating Akt/GSK-ß/Mcl-1 axis, in which mitochondria instability and caspase-9/3 activation also occurred concurrently. Pharmacological inhibition of caspase-8 and cathepsin B partly reversed tBid expression and mitochondrial damage to further attenuate DCMI-mediated cytotoxicity. Additionally, DCMI presented partial therapeutic effects in treating mesenchymal type of A549 tumor bearing nude mice through an acceleration of cancer cell death. Taken together, DCMI exerts antitumor effects via initiating the mechanisms of Akt/GSK-ß/Mcl-1 inactivation and cathepsin B/caspase-8-regulated mitochondrial death, which suggests its potential role in mesenchymal type of cancer cell therapy.

Dual-emissive carbonized polymer dots for the ratiometric fluorescence imaging of singlet oxygen in living cells.

Singlet oxygen (1O2) is a type of reactive oxygen species (ROS), playing a vital role in the physiological and pathophysiological processes. Specific probes for monitoring intracellular 1O2 still remain challenging. In this study, we develop a ratiometric fluorescent probe for the real-time intracellular detection of 1O2 using o-phenylenediamine-derived carbonized polymer dots (o-PD CPDs). The o-PD CPDs possessing dual-excitation-emission properties (blue and yellow fluorescence) were successfully synthesized in a two-phase system (water/acetonitrile) using an ionic liquid tetrabutylammonium hexafluorophosphate as a supporting electrolyte through the electrolysis of o-PD. The o-PD CPDs can act as a photosensitizer to produce 1O2 upon white LED irradiation, in turn, the generated 1O2 selectively quenches the yellow emission of the o-PD CPDs. This quenching behavior is ascribed to the specific cycloaddition reaction between 1O2 and alkene groups in the polymer scaffolds on o-PD CPDs. The interior carbon core can be a reliable internal standard since its blue fluorescence intensity remains unchanged in the presence of 1O2. The ratiometric response of o-PD CPDs is selective toward 1O2 against other ROS species. The developed o-PD CPDs have been successfully applied to monitor the 1O2 level in the intracellular environment. Furthermore, in the inflammatory neutrophil cell model, o-PD CPDs can also detect the 1O2 and other ROS species such as hypochlorous acid after phorbol 12-myristate 13-acetate (PMA)-induced inflammation. Through the dual-channel fluorescence imaging, the ratiometric response of o-PD CPDs shows great potential for detecting endogenous and stimulating 1O2in vivo.

Photoswitchable carbon-dot liposomes mediate catalytic cascade reactions for amplified dynamic treatment of tumor cells.

Most biochemical reactions that occur in living organisms are catalyzed by a series of enzymes and proceed in a tightly controlled manner. The development of artificial enzyme cascades that resemble multienzyme complexes in nature is of current interest due to their potential in various applications. In this study, a nanozyme based on photoswitchable carbon-dot liposomes (CDsomes) was developed for use in programmable catalytic cascade reactions. These CDsomes prepared from triolein are amphiphilic and self-assemble into liposome-like structures in an aqueous environment. CDsomes feature excitation-dependent photoluminescence and, notably, can undergo reversible switching between a fluorescent on-state and nonfluorescent off-state under different wavelengths of light irradiation. This switching ability enables the CDsomes to exert photocatalytic oxidase- and peroxidase-like activities in their on- (bright) and off- (dark) states, respectively, resulting in the conversion of oxygen molecules into hydrogen peroxide (H2O2), followed by the generation of active hydroxyl radicals (OH). The two steps of oxygen activation can be precisely controlled in a sequential manner by photoirradiation at different wavelengths. Catalytic reversibility also enables the CDsomes to produce sufficient reactive oxygen species (ROS) to effectively kill tumor cells. Our results reveal that CDsomes is a promising photo-cycling nanozyme for precise tumor phototherapy through regulated programmable cascade reactions.

Evaluation of chemotherapeutic response in living cells using subcellular Organelle‒Selective amphipathic carbon dots.

Monitoring of structural changes in subcellular organelles is critical to evaluate the chemotherapeutic response of cells. However, commercial organelle selective fluorophores are easily photobleached, and thus are unsuitable for real-time and long-term observation. We have developed photostable carbon-dot liposomes (CDsomes)-based fluorophores for organellar and suborganellar imaging to circumvent these issues. The CDs synthesized through a mild pyrolysis/hydrolysis process exhibit amphipathic nature and underwent self-assembly to form liposome-like structures (CDsomes). The controlled hydrophilicity or hydrophobicity-guided preparation of CDsomes are used to selectively and rapidly (<1 min) stain nucleolus, cytoplasm, and membrane. In addition, the CDsomes offer universal high-contrast staining not only in fixed cells but also in living cells, allowing real-time observation and morphological identification in the specimen. The as-prepared CDsomes exhibit excitation-dependent fluorescence, and are much more stable under photoirradiation (e.g., ultraviolet light) than traditional subcellular dyes. Interestingly, the CDsomes can be transferred to daughter cells by diluting the particles, enabling multigenerational tracking of suborganelle for up to six generations, without interrupting the staining pattern. Therefore, we believe that the ultra-photostable CDsomes with high biocompatibility, and long-term suborganellar imaging capabilities, hold a great potential for screening and evaluating therapeutic performance of various chemotherapeutic drugs.

Carbon dots with polarity-tunable characteristics for the selective detection of sodium copper chlorophyllin and copper ions.

Compared to water-soluble carbon dots (CDs) the properties and applications of hydrophobic CDs are rarely addressed. In this study, a one-pot, simple chemical oxidation approach has been applied to synthesize hydrophobic carbon dots (TO-CDs) at room temperature from triolein (TO) in concentrated sulfuric acid solution. Sodium copper chlorophyllin (SCC) quenches the fluorescence of TO-CDs by a photoinduced electron transfer process. Upon excitation at 400 nm, the fluorescence intensity of TO-CDs probe at 500 nm shows a linear response against the SCC concentration ranging from 1.0 to 10 µM, with a limit of detection (LOD) of 0.61 µM. Quantitation of SCC in flavored drinks shows percentage recovery (%R) vaues of 98-103% and relative standard deviation (RSD) values less than 6.5%. The hydrophobic TO-CDs can be converted into hydrophilic TO-CDs through hydrolysis in NaOH solution. The presence of sulfonyl groups on the hydrophilic TO-CDs enhances the coordination ability of the CDs toward Cu2+ ions, leading to fluorescence quenching which allows for the detection of Cu2+ ions with LOD of 0.21 µM and a linear range of 0.5-10 µM. The hydrophilic TO-CD probe possesses high selectivity toward Cu2+ ions (tolerance at least ten-fold comparative to other metal ions). The assay has been validated with the analysis of spiked soil samples, with %R values of Cu concentration of 97.8-99.0% and RSDs below 2.0%. The surface tunable CD probes demonstrate their potential for the rapid screening of Cu2+ ions in environmental samples and SCC in foods.

Electrocatalytic CuBr@CuO nanoparticles based salivary glucose probes.

Highly electrocatalytic cuprous halide/copper oxide nanoparticles (CuX@CunO NPs; X = Cl, Br or I; n = 1 or 2) have been fabricated on copper foils for sensitive detection of glucose. Formation of CuX@CunO NPs involves two steps- in situ electrochemical deposition of CuX on the foil and then conversion of CuX to CunO. The deposited CuX converts to CunO, leading to the generation of abundant oxygen vacancies in the CuO lattice, enhancing the number of catalytically active sites, and improving the charge transfer efficiency. Among the as-prepared electrodes, CuBr@CuO NP ones provide the highest electrocatalytic activity toward the oxidation of glucose. The electrode provides electrocatalytic activity toward the oxidation of glucose at a low overpotential of 0.25 V (vs. SCE), which is lower than that (0.40 V) of unmodified copper electrodes. The generated anodic current is proportional to glucose concentration in an alkaline medium, with a good linear range from 5.0 µM to 3.51 mM (R2 = 0.995). Its reliability has been validated by detecting the glucose concentration in saliva samples at different time intervals after a meal. The results are in good correlation with the blood glucose level determined by using a commercial blood glucose meter. Our CuBr@CuO NP electrode possesses great potential for monitoring salivary glucose to achieve the purpose of noninvasive glucose monitoring for patients with diabetes in the future.

Role of tumor endothelial marker 1 (Endosialin/CD248) lectin-like domain in lipopolysaccharide-induced macrophage activation and sepsis in mice.

Deleterious hyper-inflammation resulting from macrophage activation may aggravate sepsis and lead to lethality. Tumor endothelial marker 1 (TEM1), a type I transmembrane glycoprotein containing six functional domains, has been implicated in cancer and chronic sterile inflammatory disorders. However, the role of TEM1 in acute sepsis remains to be determined. Herein we explored the functional significance of the TEM1 lectin-like domain (TEM1D1) in monocyte/macrophage activation and sepsis using TEM1D1-deleted (TEM1LeD/LeD) transgenic mice and recombinant TEM1D1 (rTEM1D1) protein. Under stimulation with lipopolysaccharides (LPS) or several other toll-like receptor agonists, TEM1LeD/LeD macrophages produced lower levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than wild-type TEM1wt/wt macrophages. Compared with TEM1wt/wt macrophages, LPS-macrophage binding and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation were suppressed in TEM1LeD/LeD macrophages. In vivo, TEM1D1 deletion improved survival in LPS-challenged mice with reduction of circulating TNF-α and IL-6 and alleviation of lung injury and pulmonary leukocyte accumulation. In contrast, rTEM1D1 could bind to LPS and markedly suppress LPS-macrophage binding, MAPK/NF-κB signaling in macrophages and proinflammatory cytokine production. Treatment with rTEM1D1 improved survival and attenuated circulating TNF-α and IL-6, lung injury and pulmonary accumulation of leukocytes in LPS-challenged mice. These findings demonstrated differential roles for the TEM1 lectin-like domain in macrophages and soluble TEM1 lectin-like domain in sepsis. TEM1 in macrophages mediates LPS-induced inflammation via its lectin-like domain, whereas rTEM1D1 interferes with LPS-induced macrophage activation and sepsis.

High-performance FRET biosensors for single-cell and in vivo lead detection.

Forms of lead (Pb) have been insidiously invading human life for thousands of years without obvious signs of their considerable danger to human health. Blood lead level (BLL) is the routine measure used for diagnosing the degree of lead intoxication, although it is unclear whether there is any safe range of BLL. To develop a practical detection tool for living organisms, we engineered a genetically encoded fluorescence resonance energy transfer (FRET)-based Pb2+ biosensor, 'Met-lead 1.44 M1', with excellent performance. Met-lead 1.44 M1 has an apparent dissociation constant (Kd) of 25.97 nM, a detection limit (LOD) of 10 nM (2.0 ppb/0.2 µg/dL), and an enhancement dynamic ratio of nearly ~ 5-fold upon Pb2+ binding. The 10 nM sensitivity of Met-lead 1.44 M1 is five times below the World Health Organization-permitted level of lead in tap water (10 ppb; WHO, 2017), and fifteen times lower than the maximum BLL for children (3 µg/dL). We deployed Met-lead 1.44 M1 to measure Pb2+ concentrations in different living models, including two general human cell lines and one specific line, induced pluripotent stem cell (iPSC)-derived cardiomyocytes, as well as in widely used model species in plant (Arabidopsis thaliana) and animal (Drosophila melanogaster) research. Our results suggest that this new biosensor is suitable for lead toxicological research in vitro and in vivo, and will pave the way toward potential applications for both low BLL measures and rapid detection of environmental lead in its divalent form.

Monitoring the Heavy Metal Lead Inside Living Drosophila with a FRET-Based Biosensor.

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called "safe value" of the BLL seems to unreliably represent the secure threshold for children. In general, lead's underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood-brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

Carbon dots functionalized papers for high-throughput sensing of 4-chloroethcathinone and its analogues in crime sites.

Sensitive and selective assays are demanded for quantitation of new psychoactive substances such as 4-chloroethcathinone that is a π-conjugated keto compound. Carbon dots (C-dots) prepared from L-arginine through a hydrothermal route have been used for quantitation of 4-chloroethcathinone in aqueous solution and on C-dot-functionalized papers (CDFPs). To prepare CDFPs, chromatography papers, each with a pattern of 8 × 12 circles (wells), are first fabricated through a solid-ink printing method and then the C-dots are coated into the wells. π-Conjugated keto or ester compounds induce photoluminescence quenching of C-dots through an electron transfer process. At pH 7.0, the CDFPs allow screening of abused drugs such as cocaine, heroin and cathinones. Because of poor solubility of heroin and cocaine at pH 11.0, the C-dot probe is selective for cathinones. The C-dots in aqueous solution and CDFPs at pH 11.0 allow quantitation of 4-chloroethcathinone down to 1.73 mM and 0.14 mM, respectively. Our sensing system consisting of a portable UV-lamp, a smartphone and a low-cost CDFP has been used to detect cathinones, cocaine and heroin at pH 7.0, showing its potential for screening of these drugs in crime sites.

Fluorescence-tunable copper nanoclusters and their application in hexavalent chromium sensing.

Generally, metal nanoclusters are synthesized using only a single ligand. Thus, the properties and applications of these nanomaterials are limited by the nature of the ligand used. In this study, we have developed a new synthetic strategy to prepare bi-ligand copper nanoclusters (Cu NCs). These bi-ligand Cu NCs are synthesized from copper ions, thiosalicylic acid, and cysteamine by a simple one-pot method, and they exhibit high quantum yields (>18.9%) and good photostability. Most interestingly, the fluorescence intensities and surface properties of the Cu NCs can be tailored by changing the ratio of the two ligands. Consequently, the bi-ligand Cu NCs show great promise as fluorescent probes. Accordingly, the Cu NCs were applied to the inner-filter-effect-based detection of hexavalent chromium in water. A wide linear range of 0.1-1000 µM and a low detection limit (signal-to-noise ratio = 3) of 0.03 µM was obtained. The recoveries for the real sample analysis were between 98.3 and 105.0% and the relative standard deviations were below 4.54%, demonstrating the repeatability and practical utility of this assay.

Parameters affecting the synthesis of carbon dots for quantitation of copper ions.

A simple, eco-friendly, and low-cost electrochemical approach has been applied to the synthesis of carbon dots (C dots) from histidine hydrochloride in the absence or presence of halides (Cl, Br, and I) at various potentials up to 10 V. The as-formed C dots refer to C dots, Cl-C, Br-C, and I-C dots. The time-evolution UV-vis absorption and photoluminescence (PL) spectra provide more detailed information about the formation of C dots. Upon increasing the reaction time from 1 to 120 min, more and more C dots are formed, leading to increased PL intensity. The halides play two important roles in determining the formation of C dots; controlling the reaction rate and surface states. When compared to chloride and bromide, iodide has a greater effect on varying surface states and inducing PL quenching through intersystem crossing. The PL intensities of the four types of C dots all decrease upon increasing Cu2+, Hg2+, and Ag+ concentrations. In the presence of 0.8 mM I-, I-C dots compared to C dots, Cl-C dots, and Br-C dots are slightly better for quantitation of Cu2+. Fourier transform infrared spectroscopy, cyclic voltammetry, electrochemical impedance spectroscopy, and X-ray photoelectron spectroscopy results of I-C dots reveal the interactions of Cu2+ with the surface ligands (imidazole and histidine). The I-C dot probe in the presence of 0.8 mM I- is selective toward Cu2+ over the tested metal ions such as Hg2+ and Ag+. The assay provides a limit of detection of 0.22 µM for Cu2+ at a signal-to-noise ratio of 3. Practicality of this probe has been validated by the analyses of tap, lake, and sea water samples, with negligible matrix effects.

Highly sensitive sensing of hydroquinone and catechol based on ß-cyclodextrin-modified carbon dots.

In the proposed study, an efficient method for a carbon dot@ß-cyclodextrin (C-dot@ß-CD)-based fluorescent probe was developed for the analyses of catechol (CC) and hydroquinone (HQ) at trace levels in water samples. The properties of C-dot@ß-CD nanocomposites were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The sensing behaviors of C-dot@ß-CD toward CC and HQ were investigated by fluorescence spectroscopy. Based on the host-guest chemistry between C-dot@ß-CD and phenolic compounds, which can quench C-dot@ß-CD fluorescence, the prepared C-dot@ß-CD nanocomposites could be used for the sensitive and selective detection of CC or HQ across a wide linear range (0.1 to 10 µM) with detection limits of 47.9 and 20.2 nM, respectively. These results showed that the synthesized C-dot@ß-CD nanocomposite exhibited strong fluorescence and high degree of water solubility and thus, it is suitable for use as a nanoprobe for detecting CC or HQ in real water samples.

Biological Mechanisms by Which Antiproliferative Actions of Resveratrol Are Minimized.

Preclinical and clinical studies have offered evidence for protective effects of various polyphenol-rich foods against cardiovascular diseases, neurodegenerative diseases, and cancers. Resveratrol is among the most widely studied polyphenols. However, the preventive and treatment effectiveness of resveratrol in cancer remain controversial because of certain limitations in existing studies. For example, studies of the activity of resveratrol against cancer cell lines in vitro have often been conducted at concentrations in the low µM to mM range, whereas dietary resveratrol or resveratrol-containing wine rarely achieve nM concentrations in the clinic. While the mechanisms underlying the failure of resveratrol to inhibit cancer growth in the intact organism are not fully understood, the interference by thyroid hormones with the anticancer activity of resveratrol have been well documented in both in vitro and xenograft studies. Thus, endogenous thyroid hormones may explain the failure of anticancer actions of resveratrol in intact animals, or in the clinic. In this review, mechanisms involved in resveratrol-induced antiproliferation and effects of thyroid hormones on these mechanisms are discussed.

A highly selective and sensitive nanosensor for the detection of glyphosate.

A turn-off fluorescence sensor synthesized by combining copper (II) oxide and multiwall carbon nanotubes (MWCNTs) were used for measuring glyphosate based on the inhibiting the catalytic activity of the CuO/MWCNTs. This sensor was synthesized by precipitating copper ions onto the acidic MWCNTs under basic conditions; the resulting material was characterized by the transmission electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy to confirm its structure. The CuO/MWCNTs nanomaterial was found to exhibit high peroxidase-like catalytic activity toward the reduction of H2O2 to H2O and the oxidation of Amplex Red to resorufin, with a corresponding color change from pink to red and the fluorescence enhancement. However, this activity was inhibited and the fluorescence diminished when glyphosate was added to the system. Using this strategy, we applied this sensor to detect glyphosate. The results indicated that this sensor is not only highly sensitive, with a detection limit of 0.67 ppb and a linear range from 0.002 to 0.01ppm, but also exhibits good selectivity for glyphosate. When this sensor was assessed for detecting glyphosate in real water samples, recoveries of 96-107% were attained. This proposed material and method are a promising approach for rapid screening of glyphosate.

Improvement in non-specific immunity and disease resistance of barramundi, Lates calcarifer (Bloch), by diets containing Daphnia similis meal.

A 42-day study was conducted with barramundi, Lates calcarifer, to evaluate the effects of Daphnia meal derived from Daphnia similis on fish growth, immune response, and disease resistance to Aeromonas hydrophila. Three isonitrogenous (45%) and isolipid (10%) experimental diets were formulated to contain 0% (control), 5% (D5), and 10% (D10) Daphnia meal. Growth was depressed when fish were fed with the D10 diet for 42 days compared to control. However, the growth in fish fed with control and D5 diets for 42 days was not significantly different. By day 42, the leukocyte phagocytic activity and respiratory burst activity were significantly increased in D5 and D10 groups compared to control. Mx gene expression in the spleen and head kidney of fish after being injected with nerve necrosis virus was also significantly up-regulated in both groups compared to control. In an increased immune response, D5 and D10 fish had significantly higher survival rates than control after being challenged by A. hydrophila. Therefore, we suggest that a 5% Daphnia-meal diet could improve the barramundi immune response and disease resistance without a negative impact on growth.

Using spectroscopic ellipsometry to characterize and apply the optical constants of hollow gold nanoparticles.

In this paper, we report the optical constants (refractive index, extinction coefficient) of self-assembled hollow gold nanoparticle (HGN) monolayers determined through spectroscopic ellipsometry (SE). We prepared a series of HGNs exhibiting various morphologies and surface plasmon resonance (SPR) properties. The extinction coefficient (k) curves of the HGN monolayers exhibited strong SPR peaks located at wavelengths that followed similar trends to those of the SPR positions of the HGNs in solution. The refractive index (n) curves exhibited an abnormal dispersion that was due to the strong SPR extinction. The values of Deltan and kmax both correlated linearly with the particle number densities. From a comparison of the optical constant values of HGNs with those of solid Au nanoparticles (NPs), we used SE measurements to demonstrate a highly sensitive Si-based chemical sensor. HGNs display a slightly lower value of k at the SPR peak but a much higher sensitivity to changes in the surrounding medium than do solid Au NPs.